Abstract Streptococcus pneumoniae (Spn) colonizes the epithelial surface of the human nasopharynx in early childhood and remains a significant cause of respiratory illness. Globally, Spn causes 15 million cases of pneumococcal disease (PD) each year leading to approximately ?0.5 million deaths in children. Treatment of PD has been hindered by emergence of antimicrobial resistance, including the resistance to macrolides. Most pneumococcal macrolide resistance is conferred by Erm(B), the RNA methylase, and/or efflux/ribosomal protection mediated by Mef(E)/Mel on the macrolide efflux genetic assembly (Mega) element. The Mega element, related to Tn916 conjugative transposons but does not encode putative recombinases, has integrated into at least four loci in the pneumococcal chromosome, Mega classes I?IV. Molecular epidemiological studies demonstrated a higher prevalence of isolates containing the class II Mega, which is not caused by clonal expansion. Moreover, a wide array of complex Tn916 related mobile genetic elements, termed integrative and conjugative elements (ICEs), has emerged that also facilitate dissemination of macrolide resistance (both ermB and Mega) and additional antibiotic resistance markers. While acquisition of Mega and ICE-encoded resistance presumably occurs during colonization of the human nasopharynx, the efficiency and the specific mechanisms by which Mega elements and ICEs spread among pneumococci in the nasopharynx is not well understood. Our novel discovery of pneumococcal unidirectional transformation in the human nasopharyngeal biofilms has provided new insights on gene transfer in S. pneumoniae. In this proposal, using an established model of human nasopharyngeal consortial biofilms and the newly characterized uni-directional gene transfer phenomenon, we will determine the mechanisms and frequencies of macrolide resistance dissemination mediated by the Mega elements and ICEs. In Aim 1 we will evaluate whether genomic recombination hot spots or strain background, influence the recombination frequency (rF) and account for different prevalence among Mega classes. Donor strains engineered with each of the four Mega classes and defined pneumococcal clinical isolates containing the Mega classes will be conducted to assess the contribution of genomic loci and strain backgrounds, respectively. Both the rF and the specific recombination sites will be defined in recombinants. In aim 2 we will investigate whether conjugation or recombination via transformation, drives the mobilization of ICE- encoded macrolide resistant determinants. The molecular mechanism of ICE-dissemination among pneumococci will be further investigated using mutants with inactivated transposon-encoded mobilization proteins and proteins mediating competence. Identifying the horizontal dissemination mechanisms and frequencies of the two-major macrolide resistance genetic determinants will be valuable for new interventions aimed at decreasing the burden of antibiotic resistance dissemination in S. pneumoniae.